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SIRT1 alleviates high-magnitude compression-induced senescence in nucleus pulposus cells via PINK1-dependent mitophagy.

ABSTRACT: Mechanical overloading-induced nucleus pulposus (NP) cells senescence plays an important role in the pathogenesis of intervertebral disc degeneration (IVDD). The silent mating type information regulator 2 homolog-1 (SIRT1)-mediated pathway preserves the normal NP cell phenotype and mitochondrial homeostasis under multiple stresses. We aimed to investigate the role of SIRT1 in IVDD by assessing the effects of SIRT1 overexpression on high-magnitude compression-induced senescence in NP cells. High-magnitude compression induced cellular senescence and mitochondrial dysfunction in human NP cells. Moreover, SIRT1 overexpression tended to alleviate NP cell senescence and mitochondrial dysfunction under compressive stress. Given the mitophagy-inducing property of SIRT1, activity of mitophagy was evaluated in NP cells to further demonstrate the underlying mechanism. The results showed that SIRT1-overexpression attenuated senescence and mitochondrial injury in NP cells subjected to high-magnitude compression. However, depletion of PINK1, a key mitophagic regulator, impaired mitophagy and blocked the protective role of SIRT1 against compression induced senescence in NP cells. In summary, these results suggest that SIRT1 plays a protective role in alleviating NP cell senescence and mitochondrial dysfunction under high-magnitude compression, the mechanism of which is associated with the regulation of PINK1-dependent mitophagy. Our findings may provide a potential therapeutic approach for IVDD treatment.

SUBMITTER: Wang Y

PROVIDER: S-EPMC7485741 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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SIRT1 alleviates high-magnitude compression-induced senescence in nucleus pulposus cells via PINK1-dependent mitophagy.

Wang Yiyang Y Wang Haoming H Zhuo Yunyun Y Hu Yanzhu Y Zhang Zetong Z Ye Jixing J Liu Liehua L Luo Lei L Zhao Chen C Zhou Qiang Q Li Pei P

Aging 20200718 16

Mechanical overloading-induced nucleus pulposus (NP) cells senescence plays an important role in the pathogenesis of intervertebral disc degeneration (IVDD). The silent mating type information regulator 2 homolog-1 (SIRT1)-mediated pathway preserves the normal NP cell phenotype and mitochondrial homeostasis under multiple stresses. We aimed to investigate the role of SIRT1 in IVDD by assessing the effects of SIRT1 overexpression on high-magnitude compression-induced senescence in NP cells. High- ...[more]

PMID: 32687063

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Project description:Intervertebral disc degeneration (IDD) is the primary pathological mechanism that underlies low back pain. Overloading-induced cell death, especially endogenous stem cell death, is the leading factor that undermines intrinsic repair and aggravates IDD. Previous research has separately studied the effect of oxygen concentration and mechanical loading in IDD. However, how these two factors synergistically influence endogenous repair remains unclear. Therefore, we established in vitro and in vivo models to study the mechanisms by which hypoxia interacted with overloading-induced cell death of the nucleus pulposus derived stem cells (NPSCs). We found the content of HIF1A (hypoxia inducible factor 1 subunit alpha) and the number of NPSCs decreased with disc degeneration in both rats and human discs. Hence, we isolated this subpopulation from rat discs and treated them simultaneously with hypoxia and excessive mechanical stress. Our results demonstrated that hypoxia exerted protective effect on NPSCs under compression, partially through elevating macroautophagy/autophagy. Proteomics and knockdown experiments further revealed HIF1A-BNIP3-ATG7 axis mediated the increase in autophagy flux, in which HMOX1 and SLC2A1 were also involved. Moreover, HIF1A-overexpressing NPSCs exhibited stronger resistance to over-loading induced apoptosis in vitro. They also showed higher survival rates, along with elevated autophagy after being intra-disc transplanted into over-loaded discs. Jointly, both in vivo and in vitro experiments proved the anti-apoptotic effect of HIF1A on NPSCs under the excessive mechanical loading, suggesting that restoring hypoxia and manipulating autophagy is crucial to maintain the intrinsic repair and to retard disc degeneration.Abbreviations: 3-MA: 3-methyladenine; ACAN: aggrecan; ATG7: autophagy related 7; BafA1: bafilomycin A1; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CASP3: caspase 3; CCK8: cell counting kit-8; CHT: chetomin; CMP: compression; CoCl2: cobalt chloride; COL2A1: collagen type II alpha 1 chain; Ctrl: control; DAPI: 4,6-diamidino-2-phenylindole; DEP: differentially expressed protein; DiR: 1,1-dioctadecyl-3,3,3,3-tetramethyl indotricarbocyanine; ECM: extracellular matrix; FCM: flow cytometry; GD2: disialoganglioside GD 2; GFP: green fluorescent protein; GO: gene ontology; GSEA: gene set enrichment analysis; H&E: hematoxylin-eosin; HIF1A: hypoxia inducible factor 1 subunit alpha; HK2: hexokinase 2; HMOX1: heme oxygenase 1; HX: hypoxia mimicry; IDD: intervertebral disc degeneration; IF: immunofluorescence; IHC: immunohistochemistry; IVD: intervertebral disc; KEGG: kyoto encyclopedia of genes and genomes; LBP: low back pain; Lv: lentivirus; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MMP: mitochondrial membrane potential; NC: negative control; NIR: near-infrared; NP: nucleus pulposus; NPC: nucleus pulposus cell; NPSC: nucleus pulposus derived stem cell; NX: normoxia; PPI: protein-protein interactions; RFP: red fluorescent protein; SLC2A1/GLUT1: solute carrier family 2 member 1; SQSTM1/p62: sequestosome 1; TEK/TIE2: TEK receptor tyrosine kinase; TEM: transmission electron microscopy; TUBB: tubulin beta class I.

Project description:BackgroundIntervertebral disc degeneration (IDD) is a major contributor to lower back pain, however, the molecular and pathogenetic mechanisms underlying IDD are poorly understood. As a high-risk factor for IDD, compression stress was reported to induce apoptosis of nucleus pulposus (NP) cells and extracellular matrix (ECM) degradation during IDD progression. Circular RNA (circRNA) is a class of endogenous non-coding RNA (ncRNA) and has been reported to function in several diseases. However, whether and how circRNA regulates compression-induced damage of NP cells remains vague. Here, we aimed to investigate the key role of circRNA in compression loading-induced IDD.MethodsWe analysed the circRNA expression of three samples from compression-treated NP cells and three control samples using circRNA microarray assays and further investigated the circRNA involved in compression-induced damage of NP cells (circRNA-CIDN). We investigated the effects of circRNA-CIDN on compression-induced cell apoptosis and NP ECM degradation in vitro and ex vivo. We observed that circRNA-CIDN bound to miRNAs as a miRNA sponge based on luciferase and RNA immunoprecipitation (RIP) assays.FindingsCircRNA-CIDN was significantly downregulated in compression-treated human NP cells, as validated by circRNA microarray and qRT-PCR analysis, and overexpressing circRNA-CIDN inhibited compression-induced apoptosis and NP ECM degradation. Further studies demonstrated that circRNA-CIDN served as a sponge for miR-34a-5p, an important miRNA that enhanced compression-induced damage of NP cells via repressing the silent mating type information regulation 2 homolog 1 (SIRT1). CircRNA-CIDN was also verified to contain IDD development in an ex vivo IDD model.InterpretationOur results revealed that circRNA-CIDN binding to miR-34a-5p played an important role in mitigating compression loading-induced nucleus pulposus cell damage via targeting SIRT1, providing a potential therapeutic strategy for IDD treatment.FundingNational Natural Science Foundation of China (81772391, 81974348), Fundamental Research Funds for the Central Universities (2017KFYXJJ248).

Dataset Information

SIRT1 alleviates high-magnitude compression-induced senescence in nucleus pulposus cells via PINK1-dependent mitophagy.

Publications

SIRT1 alleviates high-magnitude compression-induced senescence in nucleus pulposus cells via PINK1-dependent mitophagy.

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